mmg_233_2013_genetics_genomicswikiaorg-20200214-history
Genomic Analysis of Cell Divison in C. elegans
Genomics is the study of the structure and function of an organism's genome. The development of the field of genomics was primarily the result of technological advances in the sequencing of nucleic acids, especially the development of high throughput DNA sequencers (1). This allowed for the sequencing of entire genomes quickly and effectively (1). Sequencing of entire genomes generates large amounts of data that can be used to study the biology of an organism (1). Functional genomics is the field of study that takes the information generated from genomics studies and attempts to determine how the genome and its regulation relates to an organisms biology (1). This can be done by studying gene expression, levels of mRNA transcripts, as well as protein levels (1). One particular method of studying the biological effects of gene expression is through the use of RNA mediated interference (RNAi). This method uses small interfering RNA (siRNA) or double stranded RNA (dsRNA), which are converted into siRNA by the enzyme DICER. A single siRNA strand is incorporated into the RNA-induced silencing complex (RISC) (Figure 1). RISC will then cleave any mRNA transcripts that contain complementary sequences to the incorporated siRNA strand effectively silencing gene expression (2). One study that uses this technique was conducted by Gonczy et. al. In this experiment they looked at the effects of particular genes on the development of the nematode Caenorhabditis elegans (C.elegans). Functional Genomic Analysis of Cell Division in C.elegans In the study conducted by Gonczy et. al. a functional genomic screen of chromosome III for genes involved in C.elegans development was done using RNAi. dsRNAs were generated using primer pairs, which contained T7 and T3 RNA polymerase promoters, corresponding to 2232 of a predicted 2315 open reading frames (ORF) on chromosome III. Pairs of dsRNA were injected into a developing embryo. Embryo development was monitored using time-lapse differential interference contrast (DIC) microscopy. This technique allows for high spatial and temporal resolution and slight changes in embryo development can be detected (4). The pa irs of dsRNA that gave rise to an observable phenotype were split and tested individually. Individual dsRNAs that resulted in an observable phenotype were further analyzed to ensure that they were only inhibiting expression of a single gene. This was done by making sure the primers were designed to recognize individual segments of DNA that differed in sequence with other chromosomal regions. Specifically, primers were designed to avoid regions of DNA that had 80% similarity over 200 base pairs or 90% similarity over 100 base pairs with other sections of chromosome III. In order to avoid inhibition of two genes by a single dsRNA due to mispredicted gene boundaries, dsRNAs that resulted in a phenotype were confirmed to be bind segments of DNA that were at least 1000kb from the next closest dsRNA binding site. The use of RNAi to assess the functions of genes located on chromosome III on the development of C.elegans resulted in the identification of 133 genes. Each of these genes gave rise to a phenotype observable by DIC micrscopy. These phenotypes could be broken down into 6 separate classes (Figure 2) and the 133 genes that were identified were then organized into one of the 6 classes based on what phenotype they gave rise to following RNAi (Figure 3). Overall, the researchers found that 78 dsRNA were associated with embryonic lethality, 48 were associated with a larval phenotype, 13 with an adult phenotype, and 9 resulted in sterility. Of the 133 genes identified only the function of 11 had been determined. 104 of the indentified genes had homologues in other species and the functions of which, the researchers believed, maybe determined using C.elegans. The sequence of the remaining 18 genes did not provide enough information to speculate as to their functions. Based on these findings the researchers believed that RNAi is an effective tool that can be used along with the information generated from genome sequencing to determine the biological functions of genes. Sources 1. Lee PS, Lee KH. Genomic analysis. Current Opinion in Biotechnology 2000; 11(2): 171-175. 2. Dorsett Y, Tuschl T. siRNAs: Applications in functional genomics and potential as therapeutics. Nature Reviews 2004; 3: 318-329. 3. Gonczy P, Echeverri C, Oegema K, et. al. Functional genomic analysis of cell division in C.elegans using RNAi of genes on chromosome III. Nature 2000; 16: 331-336. 4. Gonczy P et. al. Dissection of cell division processes in the one cell stage Caenorhabiditis elegans embryo by mutational analysis. Journal of Cell Biology 1999; 144: 927-946.